Abstract:
Status epilepticus (SE) is a serious neurological disorder, characterised by prolonged and/or frequent seizure activity. Following SE, a selective and delayed neuronal death (DND) occurs in limbic regions of the brain, particularly in the hippocampus. The objective of this thesis was to investigate the molecular basis of SE-induced DND in the Wistar rat hippocampus.
Following the induction of SE, moribund (i.e. dead/dying) neurons were identified by histological staining, DNA fragmentation and an increase in activated microglia. Clusterin, a glycoprotein implicated in apoptotic cell death was also observed to accumulate in the soma and axons of moribund neurons 72-144 hr following SE. Morphological evidence suggested that dying neurons exhibited many of the classical features of apoptosis (i.e. apoptotic body formation, oligo-nucleosomal DNA fragmentation and rapid phagocytosis of debris) and therefore raised the possibility that SE-induced DND might be programmed (i.e. requiring de novo protein synthesis).
To investigate this hypothesis I have examined the temporal and anatomical expression of a number of proteins which may have a critical role in SE-induced DND. The expression of the inducible transcription factors (ITFs) was examined as they couple extracellular stimulation to the transcription of late effector gene(s), resulting in long-term phenotypical changes in the neuron and therefore, they may couple SE-inducing stimulation with DND. A high correlation was shown between neurons which exhibited a delayed and prolonged ITFP expression and those which were selectively vulnerable to SE-induced DND (e.g. CA1 and CA3 pyramidal cells and dentate hilar neurons). However, administration of the protein synthesis inhibitor anisomycin following the induction of SE reduced the ITFPs expression, but resulted in an increase in SE-induced DND after 48 hr. However, the levels of brain-derived neurotrophic factor (BDNF)-like immunoreactivity were also shown to attenuate at this time after this procedure. Thus, protein synthesis inhibitors administered following SE may attenuate the level of trophic support and promote cell death.
To further investigate the role of the ITFPs in nerve cell death, etoposide, a DNA topoisomerase II inhibitor, which is known to facilitate apoptosis was infused into the hippocampus. The results suggested that a complex ITFP expression occurred which preceded nerve cell death. Moreover, this nerve cell death occurred earlier (12-24 hr) and was not anatomically selective. Furthermore, following the etoposide infusion, clusterin was expressed in the hippocampal pyramidal cells, in the dentate hilar neurons and in the dentate granule cells, however the latter exhibited the strongest BDNF-like immunoreactivity.
In summary, circumstantial evidence suggests that the ITFPs may form a critical component in the cascade of events which couple toxic stimulation to nerve cell death. However, this thesis demonstrates that the ITFPs have a complex role in DND, as although the ITFPs may be sufficient to induce DND, they may not always be necessary (e.g. in the absence of sufficient trophic support).